Composition comprising an algal proteoglycan extract and use thereof

ABSTRACT

The present application provides use of an algal proteoglycan extract and a composition comprising the same in treating or preventing tumor, pain, inflammation or diseases mediated by inflammatory factors.

TECHNICAL FIELD

The present application relates to a composition comprising an algalextract and use thereof. In particular, the present application relatesto a composition comprising an algal proteoglycan extract and use of thecomposition in treating or preventing tumor, pain, inflammation ordiseases mediated by inflammatory factors.

BACKGROUND

Pancreatic cancer is a highly malignant gastro-intestinal (GI) cancer,which has a very poor prognosis. Both diagnosis and treatment ofpancreatic cancer are difficult. Almost 90% of pancreatic cancer isductal adenocarcinoma derived from glandular epithelium. In recentyears, the morbidity and mortality of pancreatic cancer is increasingsignificantly with the five-year survival rate being <1%. It is one ofthe malignant tumors having the worst prognosis. Pancreatic cancer haslow early diagnosis rate, high surgical mortality, and low cure rate. Inrecent years, the morbidity and mortality of other cancers are alsoincreasing continuously.

The key to treatment of cancers, especially the pancreatic cancer, is toseek for an effective anti-tumor agent, which is also a focus of recentstudies.

Since the 1970s, researchers have paid increasing attention to algae,especially blue-green algae. At first, the researchers mainly focused onthe nutrition value and virulence of algae. In the meeting of the UnitedNations food and agriculture organization in 1974, spirulina wasconsidered to be an excellent food resource for people in the future.Pharmaceutical uses of algae have been appreciated since the 1980s, andthe research was mainly focused on the function of various algalextracts. The research on blue-green algae extract, especially thespirulina extract was particularly striking. The inventors in thisapplication extracted an algal proteoglycan extract from the algae (see,the Chinese Patent CN1098707C). In the present application, theinventors studied the therapeutical effect of the algal proteoglycanextract on cancers (especially pancreatic cancer), as well as on pain(such as pain associated with tumors) and inflammation (such asinflammation associated with tumors).

SUMMARY OF THE INVENTIONS

In one aspect, the present application provides use of an algalproteoglycan extract or a composition comprising the algal proteoglycanextract in the manufacture of a medicament for treating or preventingpancreatic cancer in a subject. The pancreatic cancer is a primarycancer or a metastatic cancer.

The present application further provides use of an algal proteoglycanextract or a composition comprising the algal proteoglycan extract inthe manufacture of a medicament for treating or preventing inflammationor pain. In certain embodiments, the above inflammation istumor-associated inflammation, and the above pain is tumor-associatedpain.

In another aspect, the present application provides a method fortreating or preventing pancreatic cancer in a subject, which comprisesadministering a therapeutically or prophylactically effective amount ofan algal proteoglycan extract or a composition comprising the algalproteoglycan extract to the subject. The present application furtherprovides a method for treating or preventing inflammation or pain in asubject, which comprises administering a therapeutically orprophylactically effective amount of an algal proteoglycan extract or acomposition comprising the algal proteoglycan extract to the subject.

In one aspect, the present application provides an algal proteoglycanextract or a pharmaceutical composition comprising the algalproteoglycan extract, for use in treating or preventing pancreaticcancer, pain or inflammation in a subject.

In certain embodiments, the algal proteoglycan extract and/or theextract together with one or more pharmaceutically acceptable carrierscan be formulated into pharmaceutically acceptable dosage forms,preferably be formulated into oral liquids, tablets, granules, capsules,powders, pills, syrups and injections and the like.

In certain embodiments, the algal proteoglycan extract is administeredorally, subcutaneously, intramuscularly or intraperitoneally. In apreferred embodiment, the algal proteoglycan extract is administeredorally.

In certain embodiment, the dosage of the algal proteoglycan extractadministrated to a subject is 5-500 mg/kg body weight per day. In apreferred embodiment, the dosage is 10-150 mg/kg body weight per day. Inmore preferred embodiments, the dosage is 20-120 mg/kg body weight perday.

In a further aspect, the present application provides use of an algalproteoglycan extract or a composition comprising the algal proteoglycanextract in the manufacture of a medicament for treating or preventingdiseases mediated by inflammatory factors.

In a further aspect, the present application provides a pharmaceuticalcomposition comprising an algal proteoglycan extract and one or more ofother anti-tumor agents.

In some embodiments, the pharmaceutical composition comprising an algalproteoglycan extract and one or more of other anti-tumor agents is usedto treat or prevent a tumor in a subject, wherein the tumor ispreferably pancreatic cancer, melanoma or sarcoma. The algalproteoglycan extract and other anti-tumor agents have a synergisticeffect on treatment of tumors and significantly improve thetherapeutical efficacy on tumors.

In a further aspect, the present application further provides use of apharmaceutical composition comprising an algal proteoglycan extract andone or more of other anti-tumor agents in the manufacture of amedicament for treating or preventing tumor in a subject.

In a further aspect, the present application further provides a methodfor treating or preventing a tumor in a subject, comprisingadministering a therapeutically or prophylactically effective amount ofan algal proteoglycan extract or a pharmaceutical composition comprisingthe algal proteoglycan extract and one or more of other anti-tumoragents to the subject.

In certain embodiments, the tumor is a primary cancer or a metastatictumor. In preferred embodiments, the tumor is selected from pancreaticcancer, melanoma, sarcoma, leukemia, lung cancer, esophagus cancer,lymphoma, stomach cancer, liver cancer, breast cancer, colon cancer,cutaneous squamous carcinoma or metastatic tumors thereof.

The present application further provides effects of an anti-tumorcomposition comprising the algal proteoglycan extract on anti-tumorimmunity.

The above algal proteoglycan extract is prepared according to the methoddescribed in the Chinese Patent CN1098707C, which is incorporated hereinby reference in its entirety. In particular, the method for preparing analgal proteoglycan extract comprises the following steps:

a. dissolving blue-green algae powder in 5-20 fold volume of water, andbreaking cell wall;

b. heating the resulting mixture under 60-100° C., and performingsolid-liquid separation after cooling;

c. adjusting the pH of the resulting liquid to less than 7, andperforming solid-liquid separation again;

d. adjusting the resulting liquid to a neutral pH, concentrating, anddrying if necessary.

The content of proteoglycan contained in the obtained algal proteoglycanextract is more than about 50% by weight, for example, more than about60%, more than about 65%, more than about 70%, more than about 75%, andthe like. In certain embodiments, the content of proteoglycan containedin the algal proteoglycan extract is about 70%-75% by weight.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the MTS assay protocol used for detecting the proliferationof PANC-1 cell line.

FIG. 2 shows the percentage of proliferation of PANC-1 cells after beingtreated with different concentrations of algal proteoglycan extract atday 1(D1), day 2(D2), day 3(D3), day 4(D4) and day 6(D6), compared tothe control.

FIG. 3 shows CT imaging changes of the pancreas in a patient withadvanced pancreatic cancer after taking the algal proteoglycan extractfor 3 months.

DETAILED DESCRIPTION OF INVENTIONS

The present application provides use of an algal proteoglycan extract ora pharmaceutical composition comprising the algal proteoglycan extractin the manufacture of a medicament for treating or preventing a tumorsuch as pancreatic cancer, inflammation, pain, or diseases mediated byinflammatory factors.

The “pharmaceutically acceptable carrier” disclosed herein refers to acarrier which does not interfere with the bioactivity of activeingredients, including those commonly used in the pharmaceutical field.The pharmaceutically acceptable carrier disclosed herein can be solid orliquid, including pharmaceutically acceptable excipients, buffers,emulsifiers, stabilizers, preservatives, diluents, encapsulants, fillersand the like. For example, the pharmaceutically acceptable bufferfurther comprises phosphates, acetates, citrates, borates, carbonatesand the like.

The pharmaceutical composition disclosed herein can be presented in theform of a unit dose, and can be prepared by any method well known in thepharmaceutical field. All of the methods comprise the step of combiningthe active ingredients disclosed herein with one or morepharmaceutically acceptable carriers. Generally, a composition isprepared by combining active ingredients with a liquid carrier or asolid carrier or both, followed by shaping the resultant product asrequired.

In certain embodiments, the algal proteoglycan extract disclosed hereinand/or the extract together with a pharmaceutically acceptable carriercan be formulated into pharmaceutically acceptable dosage forms, forexample, oral liquid, capsule, powder, tablet, granule, pill, syrup,injection, suppository and the like.

The algal proteoglycan extract disclosed herein is prepared according tothe method described in the Chinese Patent CN1098707C, which isincorporated herein by reference in its entirety.

In certain embodiments, the algal proteoglycan extract disclosed hereinis administered at a dosage of 5-500 mg/kg body weigh per day,preferably 20-150 mg/kg body weight per day, more preferably 20-120mg/kg body weight per day, for example, 20 mg/kg body weight per day, 30mg/kg body weight per day, 60 mg/kg body weight per day, 120 mg/kg bodyweight per day, and the like.

In another aspect, a method for treating or preventing tumors such aspancreatic cancer, inflammation, pain, or diseases mediated byinflammatory factors in a subject is provided, which comprisesadministering a therapeutically or prophylactically effective amount ofan algal proteoglycan extract or a composition comprising the algalproteoglycan extract to the subject.

In a further aspect, use of a therapeutically or prophylacticallyeffective amount of an algal proteoglycan extract or a compositioncomprising the algal proteoglycan extract in treating or preventingcancer, inflammation, pain or diseases mediated by inflammatory factorsin a subject is provided.

The term “treating” or “treatment” used herein includes inhibiting,curing, relieving or alleviating cancer, pain (such as tumor- associatedpain) or inflammation (such as tumor-associated inflammation) ordiseases mediated by inflammatory factors, as well as preventing ordelaying metastasis of a primary cancer.

In certain embodiments, the cancer can be a primary cancer, and can alsobe a metastatic cancer. In some embodiments, the cancer can bepancreatic cancer, liver cancer, colon cancer, gastric cancer, melanoma,sarcoma, leukemia, lung cancer, breast cancer, cutaneous squamouscarcinoma, esophagus cancer, lymphoma and the like.

In some embodiments, the inflammatory factors disclosed herein includebut not limited to histamine receptor H3, kallikrein-related peptidase3, kallikrein-related peptidase 13, insulin-like growth factor 2, tumornecrosis factor, leukotriene B4 receptor 2, chemokine (C-C motif) ligand4, colony stimulating factor 3 (granulocyte), dopamine receptor 4,platelet derived growth factor β polypeptide, kallikrein-relatedpeptidase 11, kallikrein-related peptidase 15, leukotriene B4 receptor,leukotriene C4 synthase, chemokine (C-C motif) ligand 2, interleukin-6,interleukin-10, interferon-α receptor 2, platelet-derived growth factorA, platelet-derived growth factor C, platelet-derived growth factor D,C-reactive protein, kallikrein-related peptidase 14, bradykinin receptorB2, tumor necrosis factor (ligand) superfamily member 11, chemokine (C-Cmotif) ligand 3, chemokine (C-C motif) ligand 7, chemokine (C-C motif)ligand 20, chemokine (C-X-C motif) ligand 2, platelet derived growthfactor polypeptide, interferon (α, β and Ω) receptor 1, interleukin 3,interleukin 9, interleukin 17B, interleukin 20, growth differentiationfactor 2, growth differentiation factor 3, macrophage migrationinhibitory factor (glycosylation-inhibiting factor), growdifferentiation factor 11, bone morphogenetic protein 2, bonemorphogenetic protein 4, thrombopoietin, kallikrein-related peptidase 4,kallikrein-related peptidase 5, bradykinin receptor B1, interferon-α6,and the like.

In some embodiments, diseases or disorders mediated by inflammatoryfactors include but not limited to chronic bladder inflammation,urethritis, vestibular vertigo, anaphylaxis, asthma (bronchial asthma,childhood asthma), liver cancer (human hepatocellular carcinoma, primaryliver cancer and the like), prostatic cancer, ovarian cancer, cervicalcancer, breast cancer, testicular cancer, colorectal cancer,osteoarthritis, primary hypertension, myocardial infarction, myocardialinfarction Parkinson's disease, Alzheimer disease, hereditaryangioedema, hemangioma (infant), cardiovascular disease, colorectalcancer, purulent meningitis, endometrial cancer, spontaneous abortion,bladder cancer, glioma, liver fibrosis, cerebral hemorrhage, non-smallcell lung cancer, acute myelocytic leukemia, cerebral infarction,pharyngeal tonsil hypertrophy, hepatitis C, multiple myeloma, systemiclupus erythematosus, pneumonia (bronchopneumonia, pediatric pneumonia,community-acquired pneumonia), ulcerative colitis, chronic obstructivepulmonary disease, pulmonary tuberculosis, Parkinson's disease,non-alcoholic fatty liver disease, hypertension (obesity-relatedhypertension, primary hypertension), autoimmune hepatitis, sclerosingcholangitis, normal-weight obesity syndrome, diabetic retinopathy,inflammatory bowel disease, hypersomnia, Behcet's disease, giant cellarteritis, squamous-cell carcinoma, basal cell carcinoma,muco-cuta-meous lymph node syndrome, colitis, allergic disease,pancreatic cancer, ovarian cancer, stomach cancer, stoke (cerebralapoplexy, ischemic stroke), drug-induced acute liver injury, chronicneutrophilic leukemia, schizophrenia, papillary thyroid carcinoma,meningiomas, diabetes mellitus (type 1 diabetes mellitus, type 2diabetes mellitus), pulmonary thromboembolism, thyroiditis, multiplemyeloma bone disease, chronic lymphocytic leukemia, chondrosarcoma, oralleukoplakia and squamous carcinomas, allergic rhinitis, chronicnasosinusitis, Listeria infection, myocarditis, multiple sclerosis,multiple myeloma, laryngocarcinoma, IgA nephropathy, viral myocarditis,colorectal cancer, psoriasis, discogenic lumbocrural pain, hepatitis B,lung cancer, pulpitis, hashimoto thyroiditis, papillary thyroidcarcinoma, pulmonary tuberculosis, leukemia, kawasaki disease, aplasticanemia, polymyositis, Hodgkin's disease, myeloid leukemia, ankylosingspondylitis, chronic nasal-rhinosinusitis with nasal polyps, diffuselarge B cell lymphoma, atopic dermatitis, chronic high altitude corpulmonale, arthritis (osteoarthritis, rheumatoid arthritis, juvenileidiopathic arthritis, spondyloarthritis), non-small cell lung cancer,allergic rhinitis, lichen planus, unstable angina pectoris, acutecoronary syndrome, periodontitis, coronary heart disease,atherosclerosis, teratocarcinoma, embryonal carcinoma, atopicdermatitis, endometrial cancer, acute pancreatitis, cervical squamouscell carcinoma, oral squamous cell carcinoma, nasopharyngeal carcinoma,esophageal squamous cell carcinoma, myelodysplastic syndrome, cerebralglioma, primary Sjogren's syndrome, anemia (aplastic anemia in children,aplastic anemia), cerebral palsy, neonatal hypoxie-ischemicencephalopathy, airway inflammation, obstructive sleep apnea hypo-pneasyndrome, gout, amygdalitis, acute respiratory distress syndrome,obsessive-compulsive disorder, non-alcoholic fatty liver disease,metabolic syndrome, neonatal sepsis, hand-foot-and-mouth disease,dermatomyositis, sepsis, encephalitis, eclampsia, cancer of biliary duct(hilar cholangiocarcinoma, diabetic macrovascular complication bile ductcarcinoma), systemic sclerosis, colon cancer, polycystic ovariansyndrome, Netherton syndrome, celiac diseases, diabetic nephropathy andthe like.

The “therapeutically effective amount” or “prophylactically effectiveamount” used herein can be determined as the case may be, and can beeasily operated by a person having ordinary skills in the art accordingto the dosage actually required, for example, being determined accordingto the body weight, age and conditions of a patient. As all of theingredients contained in the extract are nontoxic, the extract itselfcan be administered directly as required, where the composition does notcomprise a pharmaceutically acceptable carrier. Where the compositioncomprises a pharmaceutically acceptable carrier, the extract and thecarrier can be mixed by conventional methods in the pharmaceutical fieldto manufacture the required medicament.

In certain embodiments, the algal proteoglycan extract or a compositioncomprising the algal proteoglycan extract is administered orally,subcutaneously, intramuscularly or intraperitoneally. In preferredembodiments, the algal proteoglycan extract or a composition comprisingthe algal proteoglycan extract is administered orally.

The present application further provides a pharmaceutical compositioncomprising an algal proteoglycan extract and one or more of otheranti-tumor agents.

The above anti-tumor agents includes those used clinically for treatingtumors, for example, inhibitors of nucleotide synthesis (e.g.5-fluorouracil(5-FU)); anti-tumor antibiotics (e.g. adriamycin (ADM));platinum compounds (e.g. carboplatin); alkylating agents (e.g.cyclophosphamide and nitrosourea, such as carmustine); inhibitors of DNApolymerase (e.g. gemcitabine); agents interfering with protein synthesis(e.g. etoposide (VP-16)); and the like.

When the algal proteoglycan extract is administered in combination withone or more of other anti-tumor agents, the anti-tumor effect isenhanced significantly, exhibiting a synergistic effect. The synergisticeffect of the combination therapy (e.g. the combination of the algalproteoglycan extract and one or more of other anti-tumor agents) allowslower dosage administrated to a subject suffering from a certaindisorder (such as cancer or associated symptoms thereof) than that ofeach therapy alone, and/or reduced frequence of administration for thetherapy.

The term “subject” used herein refers to a mammal, including but notlimited to primates, bovine, horses, pigs, sheeps, goats, dogs, cats,and rodents such as rats and mice.

In the instant specification and claims, the terms“comprise/comprises/comprising”, “include/includes/including” and“contain/contains/containing” mean “including but not limited to”, andare not intended to exclude other parts, additives, components or steps.

It should be understood that the features, characteristics, componentsor steps described in the specific aspects, embodiments or examples inthe present application can be applied to any other aspects, embodimentsor examples described herein, unless indicated to the contrary.

The above disclosures describe the inventions in general, and thefollowing examples further illustrate the inventions. These examplesdescribed are only intended to illustrate the inventions, but notconsidered as any limitation to the inventions. Although specific termsand values are used herein, these terms and values should also beinterpreted to be illustrative, and do not limit the scope of theinventions. Unless indicated specifically, the experimental methods andtechniques in the specification are methods and techniques well known toa person skilled in the art.

EXAMPLES Example 1 Inhibitory Effect of Algal Proteoglycan Extract onPancreatic Carcinoma Example 1.1 Algal Proteoglycan Extract Inhibitedthe Proliferation of PANC-1 Pancreatic Carcinoma Cells Cell Line & CellCulture

In this study, we used a pancreatic carcinoma cell line, PANC-1. Thiscell line is cultured by AnyGenes®. Several passages were conducted toensure cell viability and allow the required amount of cells to performdifferent experiments (MTS tests and SignArrays® analysis). Informationconcerning the cell line PANC-1 was described in Table 1.

TABLE 1 Cell line Organism Type Characteristics Origin PANC-1 HomoPancreas Epithelioid ATCC ® RL1469 ™ Sapiens Carcinoma

PANC-1 cells was grown in DMEM medium (Dulbecco's Modified Eagle'sMedium; Gibco® by Life Technologies™) supplemented with 10% FBS (fetalbovine serum; Gibco® by Life Technologies™), 1% L-glutamine (200 mM;Gibco® by Life Technologies™), 1% Sodium Pyruvate (100 mM; Gibco® byLife Technologies™), 1% streptomycin-penicillin (10,000 U/mL Penicillin& 10,000 μg/mL Streptomycin; Gibco® by Life Technologies™) and incubatedat 37° C. under 5% CO₂. Cell viability will be determined by Trypan blueexclusion assay.

Preparation of Algal Proteoglycan Extract

3 kg dried spirulina powder was rinsed with 3 liters of water, andfiltered by suction. Then 30 liters of water was added to the resultingresidue, and the mixture was stirred. The mixture was heated at 88° C.for 1 hour, cooled and then filtered under reduced pressure. Thefiltrate was adjusted with hydrochloric acid to pH 3.8, then put asideovernight for centrifugal separation. The supernatant was adjusted withNa₂CO₃ solution to pH 7, and then spray-dried to give 0.599 kg algaeproteoglycan extract. The proteoglycan content in the extract was testedto be 72.3%.

The algae proteoglycan extract is weighed under sterile conditions anddissolved in 50 mg/ml PBS to give the stock solution. Aftercentrifugation at 2,500 g for 30 minutes, the supernatant was filteredthrough a 0.2 μm filter. In a MTS assay, the solution was diluted in amedium to treat the cells. In the following parts, the algalproteoglycan extract is also referred to as “Extract K”.

MTS Assay

The cell proliferation test was conducted by using the CellTiter 96®AQueous None Radioactive Cell Proliferation Assay (MTS; Promega), basedon colorimetric method for determining the number of viable cells inproliferation.

This assay kit is composed of solutions of a tetrazolium salt(3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliu m); MTS) and anelectron coupling reagent (phenazine methosulfate; PMS) to increase thechemical stability of the compound. MTS is reduced by dehydrogenaseenzymes found in metabolically active cells to a colored product solublein culture medium: the formazan. The quantity of formazan product ismeasured after 2 hours of incubation at 37° C. by the amount of 490 nmabsorbance, directly proportional to the number of living cells inculture. Finally, the half maximal inhibitory concentration (IC50) ofthe algal proteoglycan extract was determined with the experimentalmodel PANC-1 cells. All the cell culture conditions are carried out intriplicate.

Cells were seeded on 96-well culture plates (Microtest 96®; Falcon) atDay-1 (D1) (1,500 cells per well in 100 μl medium), subsequently treatedwithin the preselected concentration range. In particular, 10 differentconcentrations are selected for the algal proteoglycan extract, threewells for each concentration. Cell-free wells without addition of algalproteoglycan extract are used as control wells.

Just before adding MTS solution, cell culture medium with or withouttreatment was removed to avoid interference of the algae proteoglycanextract with this assay, and 100 μl medium was added to each well. Then,20 μl of MTS solution was added in each well and measurement of theabsorbance at 490 nm was performed after 4 hours of incubation at 37° C.under 5% CO₂. The cell culture medium with or without the algaeproteoglycan extract was changed at days D2 (Day-2) and D4 (Day-4). ODvalues at 490 nm were measured at days D0, D1, D2, D3, D4 and D6,corresponding to the global analysis of 6×96-well plates (once per day),as illustrated in FIG. 1.

Results of the Cell Proliferation Assay on the PANC-1 Cell Line

MTS results at D0

OD values at D0 were measured for testing if the previous day's cellseeding is done in a proper way and if the adapted protocol is workingon this cell line. Table 2 listed OD values at D0. We can observe thatthere was no significant statistical difference among the OD values,which indicated that the cell seeding was correct and the results wererepeatable.

TABLE 2 PANC-1 Total cell line OD value Mean value SD mean value TotalSD Day 0 0.507 0.476 0.499 0.491 0.497 0.494 0.494 0.01035 0.499 0.010630.496 0.495 0.508 0.493 0.487 0.5  0.497 0.00706 0.488 0.515 0.497 0.5110.504 0.519 0.506 0.01169

Anti-Proliferative Effect of the Algae Proteoglycan Extract on PANC-1Cell Line

OD values corresponding to different concentrations of the algaeproteoglycan extract are presented in Table 3.

TABLE 3 Extract K cell-free (mg/ml) 0 5 3.5 2.5 1.75 1 0.5 0.25 0.1 0.050.025 control Day OD 0.483 0.453  0.456  0.463  0.476  0.498  0.569 0.535  0.555  0.537  0.532  0.387  1 value 0.542 0.461  0.468  0.464 0.477  0.525  0.535  0.532  0.54  0.559  0.48  0.386  mean 0.5  0.475 0.439  0.458  0.48  0.521  0.549  0.523  0.529  0.553  0.494  0.385 value 0.508 0.463  0.454  0.462  0.478  0.515  0.551  0.530  0.541 0.550  0.502  0.386  SD 0.030 0.0111 0.0146 0.0032 0.0021 0.0146 0.01710.0062 0.0131 0.0114 0.0269 0.0010 Day OD 0.653 0.41  0.463  0.451 0.474  0.592  0.522  0.59  0.518  0.557  0.642  0.382  2 value 0.6910.42  0.506  0.441  0.433  0.61  0.499  0.611  0.533  0.605  0.597 0.382  mean 0.656 0.416  0.439  0.462  0.515  0.608  0.598  0.554 0.557  0.58  0.479  value 0.667 0.415  0.469  0.451  0.474  0.603 0.540  0.585  0.536  0.581  0.573  0.382  SD 0.021 0.0050 0.0340 0.01050.0410 0.0099 0.0518 0.0288 0.0197 0.0240 0.0842 0.0000 Day OD 0.6090.395  0.385  0.393  0.47  0.458  0.583  0.519  0.544  0.549  0.51 0.394  3 value 0.63  0.387  0.407  0.393  0.612  0.605  0.547  0.611 0.558  0.554  0.549  0.386  mean 0.52  0.407  0.401  0.392  0.527 0.462  0.612  0.626  0.523  0.481  0.473  0.383  value 0.586 0.396 0.398  0.393  0.536  0.508  0.581  0.585  0.542  0.528  0.511  0.388  SD0.058 0.0101 0.0114 0.0006 0.0715 0.0838 0.0326 0.0580 0.0176 0.04080.0380 0.0057 Day OD 0.727 0.386  0.374  0.378  0.392  0.492  0.587 0.698  0.579  0.604  0.635  0.369  4 value 0.74  0.386  0.397  0.385 0.399  0.453  0.561  0.656  0.651  0.651  0.624  0.375  mean 0.725 0.38 0.383  0.386  0.405  0.412  0.637  0.658  0.55  0.625  0.61  0.365 value 0.731 0.384  0.385  0.383  0.399  0.452  0.595  0.671  0.593 0.627  0.623  0.370  SD 0.008 0.0035 0.0116 0.0043 0.0065 0.0400 0.03860.0237 0.0520 0.0235 0.0125 0.0050 Day OD 0.619 0.373  0.361  0.377 0.375  0.376  0.508  0.627  0.637  0.532  0.558  0.359  6 value 0.6050.38  0.389  0.384  0.382  0.397  0.457  0.539  0.608  0.638  0.621 0.374  mean 0.598 0.36  0.384  0.359  0.381  0.423  0.516  0.491  0.52 0.563  0.72  0.367  value 0.607 0.371  0.378  0.373  0.379  0.399 0.494  0.552  0.588  0.578  0.633  0.367  SD 0.010 0.0102 0.0149 0.01290.0038 0.0235 0.0320 0.0690 0.0609 0.0545 0.817  0.0075

Upon calculation, the percentage of cells in proliferation versuscontrol (without Extract K) was determined. After treated with differentconcentrations of the algae proteoglycan extract each day, thepercentage of cell proliferation of PANC-1 cell line versus control waspresented in Table 4 and FIG. 2. Table 5 demonstrated the inhibitioneffect of the algae proteoglycan extract on PANC-1 cell proliferation.The inhibition percentage was calculated by comparing the survival cellnumber after treating with Extract K with the survival cell numberwithout treatment.

TABLE 4 Extract K Percentage of cell proliferation versus control (%)(mg/ml) 0 0.025 0.05 0.1 0.25 0.5 1 1.75 2.5 3.5 5 D1 100 94.82 133.79126.98 117.71 134.88 105.18 74.93 61.85 55.86 62.94 D2 100 66.98 69.7954.10 71.31 55.39 77.75 32.32 24.36 30.68 11.71 D3 100 61.91 70.64 77.5299.50 97.15 60.74 55.79 2.52 5.03 4.36 D4 100 70.18 71.19 61.96 83.3862.42 22.90 8.03 3.69 4.16 3.97 D6 100 110.66 87.67 92.11 77.15 52.7713.30 5.26 2.77 4.71 1.80

TABLE 5 Extract K Percentage of inhibition of cell proliferation versuscontrol (%) (mg/ml) 0 0.025 0.05 0.1 0.25 0.5 1 1.75 2.5 3.5 5 D1 0 5.18−33.79 −26.98 −17.71 −34.88 −5.18 25.07 38.15 44.14 37.06 D2 0 33.0230.21 45.90 28.69 44.61 22.25 67.68 75.64 69.32 88.29 D3 0 38.09 29.3622.48 0.50 2.85 39.26 44.21 97.48 94.97 95.64 D4 0 29.82 28.81 38.0416.62 37.58 77.10 91.97 96.31 95.84 96.03 D6 0 −10.66 12.33 7.89 22.8547.23 86.70 94.74 97.23 95.29 98.20

A significant anti-proliferative effect of the algae proteoglycanextract on PANC-1 cells was observed after treatment with 1.75 mg/mL,2.5 mg/mL, 3.5 mg/mL or 5 mg/mL of Extract K at D1, D2, D3, D4 or D6.For example, the percentage inhibition for 2.5 mg/mL Extract K at D1 was38.15% and the percentage inhibition for 5 mg/mL Extract K at D1 was37.06%; the percentage inhibition for 2.5 mg/mL Extract K at D2 was75.64%, and the percentage inhibition for 5 mg/mL Extract K at D2 was88.29%; and 1.75 mg/mL, 2.5 mg/mL, 3.5 mg/mL and 5 mg/mL Extract K allsignificantly inhibited the proliferation of PANC-1 cells with thepercentage inhibition more than 90%.

The IC50 value of algae proteoglycan extract is >5 mg/ml at D1, andbetween 1 and 1.75 mg/ml at D2.

The above results showed that the algal proteoglycan extract disclosedherein has a remarkable inhibitory effect on PANC-1 pancreatic carcinomacells.

Example 1.2 Clinical Case of Algal Proteoglycan Extract for Treatment ofPatients with Advanced Pancreatic Carcinoma

Patient's condition: male, born in 1927, coronary stenting in 1996 fortreatment of acute myocardial infarction; cardiac pacemaker implantationin 2003 for treatment of bradycardia; pancreatic head mass was found onDec. 3, 2013 by ultrasound detection for treatment of febrile symptom;and poorly-differentiated advanced pancreatic carcinoma was diagnosed bypercutaneous ultrasound-guided core-needle biopsy of pancreatic headmass on Dec. 26, 2013. On Jan. 14, 2014, PTCD+biliary stenting wereperformed. The algal proteoglycan extract prepared according to Example1.1 was administrated to the patient on Dec. 15, 2013, 1,350 mg/d,increased to 2,025 mg/d after 20 days. The patient received no othertreatment.

Clinical efficacy observation: the patient's condition was stabilizedafter partial remission (PR), and the overall survival was 20 months.

FIG. 3 showed CT imaging changes in the pancreas of the patient afteradministration of the algal proteoglycan extract for three months. Thearterial-phase image showed that the tumor shrank from 46×51 mm to 35×46mm after 3 months. The venous-phase imaging showed that the density ofthe central necrotic area was significantly reduced. This furtherconfirmed that the algal proteoglycan extract disclosed herein can beused for treatment of pancreatic carcinoma, and has a significantinhibitory effect on advanced pancreatic carcinoma.

Pancreatic carcinoma is a highly malignant digestive tract cancer. Thepatients with pancreatic carcinoma will have a survival of about 4months without any treatment, or 16 months after resection. However, dueto low diagnosis rate of early pancreatic carcinoma, most cases havedeveloped to the advanced stage when diagnosed, which is not suitablefor surgical treatment. The existing drug therapies cannot effectivelyprolong the survival of patients. The inventors summarized the effectsof drug therapies reported in the prior art literature (Table 6). It canbe seen from Table 6 that even with combination therapy, the overallsurvival was at most 9 months. Surprisingly, the patient with advancedpancreatic carcinoma in this Example, after taking the algalproteoglycan extract, obtained an unexpected 20-month overall survival.This demonstrates that the algal proteoglycan extract disclosed hereinis significantly superior to the existing drug therapies in thetreatment of pancreatic carcinoma patients, and this effect isunexpected.

TABLE 6 Number Researcher of cases Experimental scheme Overall survivalBerlin et al 322 Gemcitabine vs. Gemcitabine + 5-FU 5.4 vs. 6.7 months(p = 0.09) Rocha Lima et al 342 Gemcitabine vs. Gemcitabine + irinotecan6.3 vs. 6.6 months (p = 0.79) Louvet et al 313 Gemcitabine vs.Gemcitabine + oxaliplatin 7.1 vs. 9.0 months (p = 0.13) Oettle et al 565Gemcitabine vs. Gemcitabine + irinotecan 6.3 vs. 6.2 months (p = 0.79)Heinemann et al 195 Gemcitabine vs. Gemcitabine + cisplatin 6.0 vs. 7.5months (p = 0.15) Starhopoulos et al 145 Gemcitabine vs. Gemcitabine +irinotecan 6.4 vs. 6.5 months (p = 0.97) Abou-Alfa et al 349 Gemcitabinevs. Gemcitabine + exatecan 6.2 vs. 6.7 months (p = 0.52) Hermann et al319 Gemcitabine vs. Gemcitabine + 7.2 vs. 8.4 months (p = 0.234)capecitabine Cunningham et al 533 Gemcitabine vs. Gemcitabine + 7.1 vs.6.2 months (p = 0.08) capecitabine Poplon et al 832 Gemcitabine vs.Gemcitabine FDR. 4.9 vs. 6.2 months (p = 0.04) Vs Gemcitabine +oxaliplatin vs 5.7 months (p = 0.22) Colucci et al 400 Gemcitabine vs.Gemcitabine + eisplatin 7.2 vs. 8.2 months (p = 0.38)

Example 2 Therapeutic Effect of the Algal Proteoglycan Extract on LiverCancer

In this example, the effect of the algal proteoglycan extract on themedian survival time of 11 patients with hepatocellular carcinoma wasexamined.

Eleven patients with hepatocellular carcinoma were treated with thealgal proteoglycan extract prepared according to Example 1.1 (1,080mg/d), until the patient was dead or oral administration becameimpractical. The survival times of all the cases were 2˜43 months, andthe median survival time was 15.2 months for 11 patients with HCC. Thelongest survival time was 43 months, 4 patients gained a survival timeof more than 20 months, 8 patients gained a survival time of more than11 months, 9 patients gained a survival time of more than 6 months, and1 patient gained a survival time of less than 3 months. In these cases,a special patient recommended by Anhui Charity Federation, ZBFU fromAnhui Hospital, was diagnosed with primary liver cancer on Aug. 2, 2005,T2N0M0, stage II, with space occupying lesion of 3.0*3.7 cm in the rightlobe of liver; and due to splenomegaly, liver cirrhosis, left kidneystone accompanied by bleeding, no other treatment was given. Thispatient entered the study on Sep. 24, 2005 and began to take the algalproteoglycan extract prepared in accordance with Example 1.1. ZBFU diedon Apr. 6, 2009. This patient took the algal proteoglycan extractcontinuously and survived for 43 months.

The survival time of patients with liver cancer has been the mostdifficult problem in the treatment of liver cancer. Variouschemotherapies for inoperable patients can only prolong the mediansurvival time by 1-2 months. Currently, the standard medication forliver cancer is Sorafenib (trade name: NEXAVAR), with a median survivaltime of 10.7 months and 7.9 months in the treatment group and placebocontrol group, respectively. However, in this Example, after taking thealgal proteoglycan extract, the patients with liver cancer have a mediansurvival time of 15.2 months. This demonstrates that the algalproteoglycan extract disclosed herein is significantly superior to theexisting medicament in treating liver cancer.

Example 3 Therapeutic Effect of Algal Proteoglycan Extract on ColonCancer

In this example, the therapeutic effect of the algal proteoglycanextract on eight patients with colon cancer was examined.

Among these 8 patients with colon cancer, 2 patients are males and 6patients are females, aged 38 to 68, and the median age is 60.5; and thephysical condition rating ranged from 70 to 90. Two patients sufferedfrom clinical stage III colon cancer, and six patients suffered fromclinical stage IV colon cancer; and thus all cases were clinical stageIII or IV tumors. 8 cases were pathologically diagnosed (8 casesreceiving surgery).

General Condition of Subjects with Colon Cancer

Gender Male 2 cases  25.00% Female 6 cases  75.00% Age(year) Range 38-68Median age 60.5 > 65 2 cases  25.00% ≤ 65 6 cases  75.00% Physical > 804 cases  50.00% condition rating 70-80 4 cases  50.00% Median KPS 85  Diagnosis method Pathology 8 cases 100.00% Surgery + chemotherapy 3cases  37.50% History of prior Surgery + radiotherapy + 5 cases  62.50%treatment chemotherapy Recurrence & Relapse 2 cases  25.00% metastasisDistant metastasis 6 cases  75.00% Stage IIIA/IIIB 2 cases  25.00% IV 6cases  75.00%

Eight patients with colonic cancer took the algal proteoglycan extractprepared according to Example 1.1 at a dosage of 1,080 mg/d. After 56days of treatment, 1 case showed partial remission (PR), 3 cases showedstabilized disease (SD), 4 cases showed progressive disease (PD). Theefficacious rate (CR+PR/total number of cases) for treatment of theeight colon cancer cases was 12.5%, and the disease control rate(CR+PR+SD/total number of cases) was 50%.

Example 4 Therapeutic Effect of the Algal Proteoglycan Extract onGastric Cancer

The therapeutic effect of algal proteoglycan extract on patients withgastric cancer was also studied.

Among three patients with gastric cancer, all were male, aged 57 to 67,with a median age of 65. Three patients were all in advanced stage, inwhich 2 cases were clinical stage III tumors, and 1 case was clinicalstage IV tumor; and the tissue type of all cases was adenocarcinoma.Three cases were pathologically diagnosed (including 1 case receivingbiopsy and 2 cases receiving surgery).

Three patients with gastric cancer took the algal proteoglycan extractprepared according to Example 1.1 at a dosage of 1,080 mg/d. After 56days of treatment, 2 cases showed stabilized disease (SD), 1 case showedprogressive disease (PD), and the disease control rate was 66.7%.

Example 5 Therapeutic Effect of the Algal Proteoglycan Extract onLymphoma

In this example, the therapeutic effect of the algal proteoglycanextract on the patients with lymphoma was examined.

Patient's condition: 62-year-old female, diagnosed as non-Hodgkin'slymphoma NHL (diffuse large B), stage IVB (end-stage tumor). The priortreatment included 6 courses of CHOP chemotherapy, 8 courses of IMVP-16chemotherapy, and 2 courses of HDAra-c+VP-16 chemotherapy. After theabove treatment, a 3×2 cm mass was found in the left submandibularregion.

Since Mar. 2, 2003, the patients with non-Hodgkin's diffuse large Blymphoma who had relapsed after multiple chemotherapies began taking thealgae proteoglycan extracts prepared according to Example 1.1 orally ata dosage of 2,700 mg/d, three times a day. On Mar. 31, 2003, the lesionsize was reduced to 1.5×1.8 cm. The clinical efficacy was evaluated aspartial remission (PR).

The above patient with recurrent non-Hodgkin's diffuse large B lymphomaafter multiple chemotherapies achieved partial remission after 28 daysof treatment with the algal proteoglycan extract, which demonstratesthat the algal proteoglycan extract disclosed herein has a significanttherapeutic effect on lymphoma.

Example 6 Therapeutic Effect of the Algal Proteoglycan Extract onEsophageal Cancer

In this example, the therapeutic effect of the algal proteoglycanextract on patients with esophageal cancer was examined.

Two patients with esophageal cancer were enrolled in the study with thephysical condition rating of 80, and both were in advanced stage (stageIII and stage IV), with distant metastases. After treatment with thealgal proteoglycan extract prepared in accordance with example 1.1, onecase showed partial remission (PR), and one case showed stabilizeddisease (SD), as shown in the table below.

Tumor Evaluation of Assessable Patients with Esophageal Cancer

Abbreviated Serial Tumor Therapeutic No. name No. stage Tissue typeeffect 1 SXFA 3 IV Adenocarcinoma SD 2 ZSCA 2 IV Squamous cell carcinomaPR

Next, the specific conditions of the two patients were described.

Patient SXFA, male, born on Mar. 19, 1941, his condition and treatmentwere as follows:

The patient was diagnosed by tissue biopsy with poorly differentiatedadenocarcinoma of esophagus, stage IV, on Nov. 10, 2005.

On Nov. 8, 2005, the CT imaging result showed a 6 cm-long nodular upliftin the mucosa located at 40 mm from the incisors line with hemorrhagicerosions.

From Aug. 13, 2006 to Nov. 15, 2006, the patient took orally the algalproteoglycan extract prepared according to Example 1.1 at a dose of1,080 mg/d continuously, without receiving any other treatment beforeand during administration of the algal proteoglycan extract.

On Oct. 13, 2006, the CT imaging result showed 5 cm irregular fillingdefect in lower esophagus, and the lesion height was 5 mm.

The therapeutic efficacy was evaluated as stabilized disease (SD).

Patient ZSCA, male, born on Jul. 17, 1957, his condition and treatmentwere as follows:

The patient was diagnosed by the puncture and smear with esophagealsquamous cell carcinoma by the puncture and smear, stage IV, on May 15,2006.

On May 17, 2006, the CT imaging result showed 5.5×5 cm lymph node in theright clavicle.

From May 17, 2006 to Nov. 1, 2006, the patient took orally the algalproteoglycan extract prepared according to Example 1.1 at a dose of1,080 mg/d continuously, without receiving any other treatment beforeand during administration of the algal proteoglycan extract.

On Nov. 1, 2006, the CT imaging result showed 2.5×2 cm lymph node in theright clavicle.

The therapeutic efficacy was evaluated as s partial remission (PR).

The above data show that the algal proteoglycan extract disclosed hereinhas also a therapeutic effect on esophageal cancers.

Example 7 Algal Proteoglycan Extract Alleviated Tumor-Associated PainExample 7.1 Therapeutic Effect of the Algal Proteoglycan Extract onLiver Cancer Pain

Pain is a common clinical symptom of advanced liver cancer, andseriously affects patients' life quality. Strong analgesic drugs wereconventionally used to control the pain. In this Example, 18 patientswith hepatocellular carcinoma were chosen for studying the inhibitory orprophylactic effect of the algal proteoglycan extract on pain.

Eighteen patients with hepatocellular carcinoma were treated with thealgal proteoglycan extract prepared in accordance with Example 1.1 at adose of 1,080 mg/d, until the patients were dead or oral administrationbecame impractical. All cases did not take other analgesic drugs duringthe study, and 14 patients' pain was measured by a pain scale. Amongthese 14 cases, 2 cases showed no pain throughout; 7 cases showed painrelief; 5 cases maintained mild pain of 1-2 degree, without change.Typical cases included the following patients.

One patient needed to take strong morphine analgesic drugs when enteringthe study, and the analgesic drugs were not needed shortly afteradministration of the algae proteoglycan extract. Another patient had ahuge tumor (8×8.5 cm) detected by imaging examination, but no painsymptom appeared in the patient after administration of the algaeproteoglycan extract.

The above data showed that the algal proteoglycan extract disclosedherein has a significant inhibitory and ameliorating effect on pain.

Pain assessment scales used herein are as follows:

Rating of the Patients' Life Quality

A. Appetite: B. Sleep: {circle around (1)}. Food intake almost {circlearound (1)}. Difficult to fall impossible; asleep; {circle around (2)}.Food intake < 1/2 of normal {circle around (2)}. Very poor sleep;intake; {circle around (3)}. Food intake =1/2 of normal {circle around(3)}. Poor sleep; intake; {circle around (4)}. Food intake slightlyreduced; {circle around (4)}. Slightly poor sleep; {circle around (5)}.Food intake remains normal. {circle around (5)}. Roughly normal. C.Pain: D. Tumor-related symptoms {circle around (1)}. Severe pain, withpassive (except for pain): position, intolerable; {circle around (1)}.Severe, intolerable; {circle around (2)}. Severe pain; {circle around(2)}. Severe; {circle around (3)}. Moderate pain; {circle around (3)}.Moderate; {circle around (4)}. Mild pain; {circle around (4)}. Mild;{circle around (5)}. Painless. {circle around (5)}. No. E. PS (ECOG):{circle around (1)}. IV; {circle around (2)}. III; {circle around (3)}.II; {circle around (4)}. I; {circle around (5)}. 0.

Example 7.2 Therapeutic Effect of the Algal Proteoglycan Extract onPancreatic Carcinoma Pain

The inhibitory effect of the algal proteoglycan extract on pain in 24patients with pancreatic cancer and pain symptoms were also studied. 24patients with pancreatic carcinoma enrolled in the study were treatedwith the algal proteoglycan extract prepared in accordance with Example1.1 at a dose of 2,025 mg/d, until the patients were dead or oraladministration became impractical. The pain symptoms in 24 patients weresignificantly alleviated, as measured by pain assessment scale.

Example 8 Inhibition Effect of the Algal Proteoglycan Extract onInflammation

Tumor-promoting inflammation is considered to be one of thecharacteristics of cancer. Chronic inflammatory diseases can increasethe risk of certain cancers and a number of epidemiological evidencesuggests that NSAIDs, especially aspirin, are potent chemicalprophylaxis agents. There are many different inflammatory cells in thetumor microenvironment; and targeting these factors in hereditary,transplantable and inducible mouse models of cancer can significantlyreduce the onset, development and spread of tumor diseases. Therefore,this complex network of inflammation provides a target for malignantdisease prevention and treatment. This Example examined the effect ofthe algal proteoglycan extract on the expression of inflammatorymediators in cancer cells.

Example 8.1 Regulatory Effect of the Algal Proteoglycan Extract onInflammatory Mediators Expression in Pancreatic Cancer Model

According to the protocols described in Example 1, the algalproteoglycan extract was prepared and the PANC-I pancreatic carcinomacell line was cultured.

The cells were seeded on 100 mm petri dishes (Corning®) (250,000 cellsper petri dish in 16 ml medium) at Day 1. PANC-1 cells were treated with5 mg/ml algae proteoglycan extract or PBS-containing medium (control) atDay 2. Cells were divided into three groups: the control group, the3-hour treatment group and the 18-hour treatment group, each group beingrepeated twice.

By using the SignArrays® system by AnyGenes®, the expression of factorsassociated with six qPCR signaling pathways (SignArrays® by AnyGenes®)including angiogenesis, inflammation, interferon & immune system,cytokines, oxidative stress, and HLA systems was studied. Table 7 listedthe number of factors whose expression was significantly down-regulated(at least 1-fold or more) compared to the control sample, among 76inflammatory mediators examined. The fold change of some exemplaryfactors which were down-regulated compared to the control was listed inTable 8.

TABLE 7 Number of factors Number of factors Treatment with significantlydown- down-regulated time regulated expression 2-fold or more  3 hours46 12 18 hours 55 13

TABLE 8 Fold change (3-hr Name of factors Symbols or 18-hr treatment)Histamine receptor H3 HRH3 3.46 kallikrein-related peptidase 3 KLK3 3.07kallikrein-related peptidase 13 KLK13 3.07 Insulin-like growth factor 2IGF2 3.66 Tumor necrosis factor TNF 2.8 Leukotriene B4 receptor 2 LTB4R22.62 Chemokine (C-C motif) ligand 4 CCL4 2.48 Colony stimulating factor3 (granulocyte) CSF3 2.79 Dopamine receptor 4 DRD4 4.13 Platelet-derivedgrowth factor beta PDGFB 2.49 polypeptide kallikrein-related peptidase11KLK11 3.22 kallikrein-related peptidase15 KLK15 1.15 Leukotriene B4receptor LTB4R 2.04 Leukotriene C4 synthase LTC4S 2.38 Chemokine (C-Cmotif) ligand 2 CCL2 1.39 Interleukin 6 IL6 1.41 Interleukin 10 IL101.46 Interferon (α) receptor 2 IFNAR2 1.08 Platelet-derived growthfactor A PDGFA 1.15 Platelet-derived growth factor C PDGFC 1.2Platelet-derived growth factor D PDGFD 1.97 C-reactive protein CRP 1.93

From the above results, it can be seen that the algal proteoglycanextract disclosed herein can regulate the expression of variousinflammatory mediators and cytokines in PANC-1 pancreatic carcinomacells, and has a significant inhibitory effect on the expression ofmultiple inflammatory factors.

Example 8.2 Regulatory Effect of the Algal Proteoglycan Extract onExpression of Inflammatory Mediators in Liver Cancer and Lung CancerModels

In the present study, HuH7 hepatocellular carcinoma cell lines and A549lung adenocarcinoma cell lines were used. The two cell lines werecultured by AnyGenes®.

HuH7 cells were grown in DMEM medium supplemented with 10% FBS (fetalbovine serum, Gibco® by Life Technologies™), 1% L-glutamine (200 mM;Gibco® by Life Technologies™), 1% sodium pyruvate (100 mM; Gibco® byLife Technologies™) and 1% streptomycin-penicillin (10,000 U/mLpenicillin & 10,000 μg/mL streptomycin; Gibco® by Life Technologies™),and incubated in 37° C. under 5% CO2. Cell viability was determined bytrypan blue exclusion assay.

A549 cells were grown in RPMI medium supplemented with 10% FBS (fetalbovine serum; Gibco® by Life Technologies™), 1% L-glutamine (200 mM;Gibco® by Life Technologies™), 1% sodium pyruvate (100 mM; Gibco® byLife Technologies™), 1% streptomycin-penicillin (10,000 U/mL Penicillin& 10,000 μg/mL Streptomycin; Gibco® by Life Technologies™), andincubated in 37° C. under 5% CO2. Cell viability was determined bytrypan blue exclusion assay.

The algal proteoglycan extract was prepared according to the protocoldescribed in Example 1. The expression of the relevant factors wasexamined according to the method described in Example 3.1. Table 9listed the number of inflammatory mediators and cytokines examined inA549 cells and in HuH7 cells, and the number of factors whose expressionwas significantly down-regulated (at least 2-fold or more) compared tothe control samples after 6 or 12 hours of treatment. Table 10 showedthe fold change of some exemplary factors which were down-regulatedcompared to the control.

TABLE 9 Number of Number of factors down- tested factors regulated2-fold or more A549 cell 63 54 HuH7 cell 29 28

TABLE 10 Fold change Fold change Name of factors Symbols (in A549 cells)(in HuH7 cells) kallikrein-related peptidase 11 KLK11 11.70kallikrein-related peptidase 14 KLK14 7.27 Bradykinin receptor B2 BDKRB29.73 Tumor necrosis factor TNF 18.69 Tumor necrosis factor (ligand)TNFSF11 5.07 superfamily member 11 Chemokine (C-C motif) ligand 3 CCL38.25 8 Chemokine (C-C motif) ligand 7 CCL7 11.64 12.51 Chemokine (C-Cmotif) ligand 20 CCL20 6.27 4.71 Chemokine (C-X-C motif) Ligand 2 CXCL213.77 Platelet-derived growth factor beta PDGFB 2.75 polypeptideInterferon (α, β and Ω) receptor 1 IFNAR1 5.01 Interleukin 3 IL3 10.5Interleukin 9 IL9 13.88 Interleukin 17B IL17B 15.21 Interleukin 20 IL2045.27 Growth differentiation factor 3 GDF3 36.53 Macrophage migrationinhibitory MIF 5.89 factor (glycosylation-inhibitory factor) Growthdifferentiation Factor 2 GDF2 1172.65 Growth differentiation factor 11GDF11 8.42 Bone morphogenetic protein 2 BMP2 5.67 2.72 Bonemorphogenetic protein 4 BMP4 2.36 6.04 Thrombopoietin THPO 6.29kallikrein-related peptidase 4 KLK4 4.48 kallikrein-related peptidase 5KLK5 6.92 Platelet-derived growth factor C PDGFC 2.83 2.52Platelet-derived growth factor D PDGFD 3.15 kallikrein-related peptidase15 KLK15 2.32 C-reactive protein CRP 25.15 12.15 Bradykinin receptor B1BDKRB1 2.71 Interleukin α6 IFNA6 27.34

The results in Examples 8.1 and 8.2 show that the algal proteoglycanextract disclosed herein appears to be a major modulator for regulatingexpression of inflammatory mediators and cytokines in a variety ofcancer models and can significantly inhibit expression of variousproinflammatory cytokines, which demonstrates that the algalproteoglycan extract disclosed herein is capable of inhibiting theoccurrence and progression of inflammation, in particulartumor-associated inflammation.

In addition, Table 11 below shows the corresponding relationship betweeninflammatory factors and diseases mediated by the factors, from which itcan be seen that the inflammatory mediators and cytokines that areregulated by the algal proteoglycan extract disclosed herein can mediatethe development of various diseases. Therefore, the algae proteoglycanextracts capable of down-regulating these inflammatory factors can beused to treat or prevent a variety of diseases and disorders associatedwith the inflammatory factors, including but not limited to the diseasesor disorders listed in table 11.

TABLE 11 Name of Factors Corresponding Diseases Histamine receptorH3(HRH3) chronic bladder inflammation, urethritis, vestibular vertigo,anaphylaxis, asthma, liver cancer, colon cancer kallikrein-relatedpeptidase 3 Prostate cancer, colorectal cancer, testicular cancer (KLK3)kallikrein-related peptidase 11 Prostate cancer, gastric cancer (KLK11)kallikrein-related peptidase 14 colorectal cancer, ovarian cancer,cervical cancer, (KLK14) Insulin-like growth factor 2(IGF2) Hemangioma(infant), cardiovascular disease, colorectal cancer, purulentmeningitis, endometrial cancer, human hepatocellular carcinoma, primaryliver cancer, spontaneous abortion, bladder cancer, schizophrenia, brainglioma, liver cancer, gastric cancer, liver fibrosis, colorectal cancer,cerebral hemorrhage, non-small cell lung cancer, hepatitis B, breastcancer, acute myeloid leukemia Tumor necrosis factor (TNF) cerebralinfarction, pharyngeal tonsil hypertrophy, hepatitis C, multiplemyeloma, systemic lupus erythematosus, rheumatoid arthritisinflammation, mycoplasma pneumonia, ulcerative colitis, bronchialasthma, chronic obstructive pulmonary disease, tuberculosis, Parkinson's disease, non-alcoholic fatty liver disease, obesity-typehypertension, juvenile idiopathic arthritis, allergic rhinitis,pancreatic cancer, non-small cell lung cancer, gastric cancer,hepatocellular carcinoma, coronary heart disease, autoimmune hepatitis,sclerosing cholangitis, breast cancer, normal-weight obesity syndrome,diabetic retinopathy, hepatitis C, psoriasis, cervical cancer,spondyloarthritis, inflammatory bowel disease, lupus erythematosus,hypersomnia, lung cancer, Behcet's disease, giant cell arteritisLeukotriene B4 receptor Non-small cell lung cancer, squamous cellcarcinoma, basal 2(LTB4R2) cell carcinoma, psoriasis vulgaris,muco-cuta-meous lymph node syndrome, colitis, rheumatoid arthritis,juvenile idiopathic arthritis, allergic disease, airway inflammation,pancreas cancer, leukemia, ovarian cancer, allergic disease, bronchialasthma, gastric cancer, ischemic stroke Chemokine (C-C motif) ligand 4drug-induced acute liver injury (CCL4) Colony stimulating factor 3Chronic neutrophilic leukemia, systemic lupus (granulocyte)(CSF3)erythematosus Dopamine receptor 4 (DRD4) Schizophrenia Platelet-derivedgrowth factor beta Papillary thyroid carcinoma, bronchial asthma,meningioma, polypeptide (PDGFB) type-2 diabetes, pulmonarythromboembolism Bradykinin receptor B2 Osteoarthritis, hypertension,myocardial infarction, (BDKRB2) Parkinson's disease, osteoarthritis,Alzheimer's disease, hereditary angioedema Tumor necrosis factor(ligand) Thyroiditis, papillary thyroid carcinoma superfamily member11(TNFSF11) Chemokine (C-C motif) ligand 3 Chronic hepatitis B,tuberculosis, multiple myeloma bone (CCL3) disease, chronic lymphocyticleukemia, systemic lupus erythematosus, chondrosarcoma Chemokine (C-Cmotif) ligand Oral leukoplakia and squamous cell carcinoma, allergic7(CCL7) rhinitis, chronic sinusitis, Listeria infection, ulcerativecolitis, myocarditis and multiple sclerosis, multiple myeloma, gastriccancer, chronic hepatitis B, prostate cancer, laryngeal cancer Chemokine(C-C motif) ligand 20 IgA nephropathy, liver cancer, viral myocarditis,chronic (CCL20) obstructive pulmonary disease, colorectal cancer,psoriasis, gastric cancer, laryngeal squamous cell carcinoma, discogeniclumbocrural pain, chronic hepatitis B, systemic lupus erythematosus,lung cancer, breast cancer, brain stroke, colon cancer, pulpitis,non-small cell lung cancer, esophageal squamous cell carcinoma Chemokine(C-X-C motif) Prostate cancer, colorectal cancer, Hashimoto'sthyroiditis, Ligand 2 (CXCL2) papillary thyroid carcinoma, tuberculosisInterleukin 3 (IL3) Leukemia, colorectal cancer, Kawasaki disease,aplastic anemia, liver cancer Interferon (α, β and Ω) receptor 1 Chronichepatitis B, chronic hepatitis C, gastric cancer, (IFNAR1) systemiclupus erythematosus, polymyositis, polydermatomyositis Interleukin 9(IL9) Hodgkin's disease, myeloid leukemia, hepatitis B, ankylosingspondylitis, chronic nasal-rhinosinusitis with nasal polyps, diffuselarge B-cell lymphoma, atopic dermatitis, chronic high altitude corpulmonale, lupus erythematosus, arthritis, rheumatoid disease, bronchialasthma, chronic obstructive pulmonary disease Interleukin 17B (IL17B)Laryngeal squamous cell carcinoma, multiple myeloma, non-small cell lungcancer, gastric cancer, childhood asthma, colon cancer, pancreaticcancer Interleukin 20 (IL20) Allergic rhinitis, chronic obstructivepulmonary disease, psoriasis, rheumatoid arthritis, bronchial asthma,lichen planus, unstable angina, acute coronary syndrome, periodontitisGrowth differentiation factor 3 Prostate cancer, breast cancer,teratocarcinoma, embryonal (GDF3) carcinoma Macrophage migrationinhibitory Ovarian cancer, coronary heart disease, atherosclerosis,factor (glycosylation-inhibitory atopic dermatitis, endometrial cancer,cerebral infarction, factor)(MIF) acute pancreatitis, cervical squamouscell carcinoma, gastric cancer, colorectal cancer, rheumatoid arthritis,oral squamous cell carcinoma, liver cancer, colon cancer, non-small celllung cancer, diabetes, nasopharyngeal carcinoma, bladder cancer,esophageal squamous cell carcinoma Bone morphogenetic protein (BMP)Brain glioma, liver cancer, colorectal cancer Thrombopoietin (THPO)Acute cerebral infarction, primary Sjogren's syndrome, childhoodaplastic anemia, aplastic anemia, hypertension, coronary heart disease,cerebral palsy, neonatal hypoxic-ischemic encephalopathy, hepatitis Bkallikrein-related peptidase Prostate cancer, ovarian cancer, thyroidcancer, breast 15(KLK15) cancer, celiac disease, childhood leukemiaLeukotriene C4 synthase (LTC4S) Ischemic stroke, asthma, obstructivesleep apnea hypopnea syndrome, lupus erythematosus, cardiovasculardisease Chemokine (C-C motif) ligand 2 Parkinson's disease, breastcancer, leukemia, prostate (CCL2) cancer, hypoxic-ischemic brain damage,colorectal cancer, hepatocellular carcinoma, gout, tonsillitis, gastriccancer, acute respiratory distress syndrome, type-2 diabetes, coronaryheart disease, obsessive compulsive disorder, rheumatoid arthritis, kneeosteoarthritis, chronic kidney disease, ischemic stroke, nonalcoholicfatty liver disease Interleukin 6 (IL6) Heart failure disease; pulmonaryinfection; metabolic syndrome; type-2 diabetes; coronary heart disease;fatty liver; neonatal sepsis; hand-foot-and-mouth disease; chronicobstructive pulmonary disease; liver cancer; rheumatoid arthritis;hepatitis B; transient ischemic attack; cervical cancer; acute coronarysyndromes; endometrial cancer; lung cancer; ischemic stroke; prostatecancer; lupus erythematosus; esophageal squamous cell carcinoma;multiple sclerosis; dermatomyositis; nasopharyngeal carcinoma;cardiovascular disease; prostate cancer, inflammatory diseases of thenervous system Interleukin 10 (IL10) Inflammatory bowel disease;rheumatoid arthritis; psoriasis; gastric cancer; coronary heart disease;periodontal disease; cardiovascular disease; asthma; type-1 diabetes;schizophrenia; chronic obstructive pulmonary disease; sepsis; atopicdermatitis; type-2 diabetes; acute coronary syndrome; hepatitis B; lupuserythematosus; hand-foot-and-mouth disease; encephalitis; cervicalcancer; eclampsia; tuberculosis Interferon (α) receptor 2(IFNAR2)Hepatitis C, hepatitis B Platelet derived growth factor A Hilarcholangiocarcinoma, systemic sclerosis, colorectal (PDGFA) cancer,ulcerative colitis, cervical squamous cell carcinoma, nasopharyngealcarcinoma Platelet derived growth factor C Breast cancer (PDGFC)Platelet derived growth factor D Breast cancer, gastric cancer,colorectal cancer, liver cancer, (PDGFD) coronary heart disease,diabetic macrovascular complications cholangiocarcinoma, esophagealsquamous cell carcinoma, coronary atherosclerotic heart disease, ovariancancer, cerebral hemorrhage, prostate cancer, ischemic stroke C-reactiveprotein(CRP) Childhood pneumonia, chronic obstructive pulmonary disease,coronary heart disease, asthma, polycystic ovary syndrome, hypertension,obstructive sleep apnea hypopnea syndrome, neonatal sepsis, depression,Kawasaki disease, community-acquired pneumonia, diabetes nephropathy,rheumatoid arthritis, type-2 diabetes, cerebral infarction,pancreatitis, sepsis, colon cancer, schizophrenia, renal cell carcinoma,myocardial infarction, atherosclerosis, cerebrovascular disease,ischemic cardio-cerebrovascular disease, metabolic syndromekallikrein-related peptidase Endometrial cancer, breast cancer, ovariancancer, 4(KLK4) colorectal cancer kallikrein-related peptidase Breastcancer, colorectal adenocarcinoma, rectal cancer, 5 (KLK5 ) ovariancancer, Netherton's syndrome, atopic dermatitis Bradykinin receptorB1(BDKRB1) Cervical cancer, bladder cancer, endometrial cancer,gallbladder cancer Interleukin α6 (IFNA6) Hepatitis B

Example 9 Synergistic Effect of Algal Proteoglycan Extract inCombination with Chemotherapeutic Agents on the Treatment of Cancer

The experimental animals used in this study were female C57 mice (18-22g) or female KM mice (18-22 g), which were provided by ShanghaiExperimental Animal Center of the Chinese Academy of Sciences,Certificate No.: 005.

Tumors, including B-16 melanoma and S-180 sarcoma, were provided byShanghai Institute of Materia Medica of the Chinese Academy of Sciences.

Carboplatin was purchased from Shandong Qilu Pharmaceutical Factory.Adriamycin was purchased from Zhejiang Haimen Pharmaceutical Factory.Etoposide was purchased from Pudong Pharmaceutical Factory of YadongPharmaceutical Company of Shanghai Pharmaceutical Industry ResearchInstitute. 5-fluorouracil was purchased from Shanghai Xudong HaipuPharmaceutical Co., Ltd. Cyclophosphamide was purchased from ShanghaiHualian Pharmaceutical Co., Ltd. The rest chemotherapy drugs werepurchased from Beijing Kerui Biological Engineering Co., Ltd.

Example 9.1 Therapeutic Effect of Algal Proteoglycan Extract inCombination with Other Chemotherapeutic Agents on Melanoma ExperimentalScheme

Female C57 mice (18-22 g) and well-grown B16 melanoma on 7-11 days wereused. The tumor tissue was made into cell suspension. The mice wereinoculated subcutaneously in the right axilla with the cell suspension,about 4.5-5×10⁶ cells/mouse. After 24 hours, the mice were randomlydivided into different groups and housed in separate cages (n=6). Algalproteoglycan extract was administered orally, and carboplatin,doxorubicin or CTX were administered intraperitoneally, once a day. Theadministration began on the second day and lasted for seven days, withdifferent doses. The blank control group was given normal saline (NS).The animals were sacrificed and weighed (g body weight) 24 hours afterdrug withdrawal. The tumors were excised and weighed. The average tumorweight was calculated for each group. According to the followingformula, the tumor inhibition rate was calculated and T test wasconducted.

${{Tumor}\mspace{14mu} {inhibition}\mspace{14mu} {rate}} = {\frac{A - B}{A} \times 100\%}$

A=average tumor weight (g) of blank control group,

B =average tumor weight (g) of therapeutic group

Dosage

According to the results of preliminary experiments, the dose of algalproteoglycan extract (hereinafter referred to as algal proteoglycan) wasset at 30 mg/kg and 60 mg/kg, the doses of carboplatin were 4 mg/kg and8 mg/kg, the dose of doxorubicin was 1 mg/kg, and CTX was administeredat 7.5 mg/Kg.

Experimental Results

TABLE 12 Therapeutic Effect of Algal Proteoglycan (30 mg/kg) Combinedwith Carboplatin on Melanoma Number of Body Weight Tumor Animals (g)Weight Tumor Route of in the in the in the in the (g) Inhibition GroupDose Administration Beginning End Beginning End x ± SD Rate (%) P ValueNS — 20 20 17.8 18.9 1.49 ± 0.50 — — Carboplatin  4 mg/kg i.p 10 9 18.016.7 1.07 ± 0.88 28.2 >0.05 Algal proteoglycan 30 mg/kg p.o 10 10 17.917.5 1.30 ± 0.65 12.8 >0.05 Carboplatin + 4 mg + i.p + p.o 10 10 17.618.1 0.91 ± 0.62 38.9 <0.01 Algal proteoglycan 30 mg/kg

TABLE 13 Therapeutic Effect of Algal Proteoglycan (60 mg/kg) Combinedwith Carboplatin on Melanoma Number of Body Weight Tumor Animals (g)Weight Tumor Route of in the in the in the in the (g) Inhibition GroupDose Administration Beginning End Beginning End x ± SD Rate (%) P ValueNS — 20 18 20.5 23.7 1.43 ± 0.51 — — Carboplatin  4 mg/kg i.p 10 9 20.323.2 1.23 ± 0.37 14.0 >0.05 Algal proteoglycan 60 mg/kg p.o 10 10 20.522.6 1.00 ± 0.36 30.1 <0.01 Carboplatin + 4 mg + i.p + p.o 10 9 20.523.3 0.85 ± 0.50 40.6 <0.01 Algal proteoglycan 60 mg/kg

TABLE 14 Therapeutic Effect of Algal Proteoglycan Combined withAdriamycin on Melanoma Number of Body Weight Tumor Animals (g) WeightTumor Route of in the in the in the in the (g) Inhibition Group DoseAdministration Beginning End Beginning End x ± SD Rate (%) P Value NS —40 40 20.6 26.4 3.37 ± 1.27 — — Adriamycin  1 mg/kg i.p 10 10 20.8 24.81.97 ± 0.84 41.5 <0.01 Algal proteoglycan 30 mg/kg p.o 10 10 20.5 25.42.84 ± 0.80 15.7 >0.05 Adriamycin + 1 mg + i.p + p.o 10 10 20.7 23.21.53 ± 0.54 54.6 <0.01 Algal proteoglycan 30 mg/kg

The above results showed that, compared with the monotherapy, theantitumor effect of algal proteoglycan combined with carboplatin ordoxorubicin was significantly enhanced, but the toxicity was notincreased. Taking algal proteoglycan at a dose of 30 mg/kg for example,although algal proteoglycan (30 mg/kg) did not effectively inhibitmelanoma growth alone (P>0.05), it can significantly enhance the effectof other antitumor drugs. For example, when algal proteoglycan (30mg/kg) was used in combination with carboplatin, the antitumor effect ofcarboplatin was significantly increased (with tumor inhibition ratebeing 38.9%), compared to the monotherapy of carboplatin (P<0.01). Whenalgal proteoglycan (30 mg/kg) was used in combination with doxorubicin,the tumor inhibition rate of doxorubicin was significantly increased by13.1%. When carboplatin was combined with algal proteoglycan (60 mg/kg),the inhibition rate of carboplatin was significantly increased by 26.6%.

The above results show that the algal proteoglycan extract disclosedherein can enhance the therapeutic effect of chemotherapeutic agentssuch as carboplatin and doxorubicin on transplanted tumor of B16melanoma in mice, demonstrating that the algae proteoglycan extract andthe commonly-used chemotherapeutic agents have a synergistic effect inthe treatment of melanoma.

Example 9.2 Therapeutic Effect of Algal Proteoglycan Extract inCombination with Other Chemotherapeutic Agents on S-180 Sarcoma in MiceExperimental Scheme

Female KM mice (18-22 g) and well-grown S-180 melanoma on 7-11 days wereused. The tumor tissue was made into cell suspension. The mice wereinoculated subcutaneously in the right axilla with the cell suspension,about 4.5-5×106 cells/mouse. After 24 hours, the mice were randomlydivided into different groups and housed in separate cages (n=6). Algalproteoglycan extract (hereinafter referred to as algal proteoglycan) wasadministered orally; and cyclophosphamide, carboplatin, 5-fluorouraciland etoposide were administered intraperitoneally, once a day. Theadministration began on the second day and lasted for seven days, withdifferent doses. The blank control group was given normal saline(NS).The animals were sacrificed and weighed (g body weight) 24 hoursafter drug withdrawal. The tumors were excised and weighed. The averagetumor weight was calculated for each group. According to the followingformula, the tumor inhibition rate was calculated and T test wasconducted.

According to the formula below, the tumor inhibition rate was calculatedand T test was conducted.

${{Tumor}\mspace{14mu} {inhibition}\mspace{14mu} {rate}} = {\frac{A - B}{A} \times 100\%}$

A=average tumor weight (g) of blank control group,

B=average tumor weight (g) of therapeutic group

Experimental Results

TABLE 15 Therapeutic Effect of Algal Proteoglycan Combined withCyclophosphamide on S-180 Sarcoma in Mice Number of Body Weight TumorAnimals (g) Weight Tumor Route of in the in the in the in the (g)Inhibition Group Dose Administration Beginning End Beginning End x ± SDRate (%) P Value NS — 20 19 22.7 25.5 1.92 ± 0.51 — — Cyclophosphamide7.5 mg/kg i.p 10 10 22.8 24.9 1.11 ± 0.24 42.2 <0.01 Algal proteoglycan 60 mg/kg p.o 10 9 23.0 23.4 1.24 ± 0.49 35.4 <0.01 Cyclophosphamide +7.5 mg + i.p + p.o 10 9 22.9 25.4 0.91 ± 0.23 52.6 <0.01 algalproteoglycan 60 mg/kg

TABLE 16 Therapeutic Effect of Algal Proteoglycan Combined withCarboplatin on S-180 Sarcoma in Mice Number of Body Weight Tumor Animals(g) Weight Tumor Route of in the in the in the in the (g) InhibitionGroup Dose Administration Beginning End Beginning End x ± SD Rate (%) PValue NS — 30 30 18.7 30.4 1.82 ± 0.69 — — Carboplatin  4 mg/kg i.p 1010 18.6 26.4 1.68 ± 0.53 7.7 >0.05 Algal proteoglycan 20 mg/kg p.o 10 1018.7 27.8 1.75 ± 0.48 3.8 >0.05 Carboplatin + 4 mg + i.p + p.o 10 1018.8 24.7 1.22 ± 0.54 33.0 <0.05 algal proteoglycan 20 mg/kg

TABLE 17 Therapeutic Effect of Algal Proteoglycan Combined withEtoposide on S-180 Sarcoma in Mice Number of Body Weight Tumor Animals(g) Weight Tumor Route of in the in the in the in the (g) InhibitionGroup Dose Administration Beginning End Beginning End x ± SD Rate (%) PValue NS — 30 30 18.7 30.4 1.82 ± 0.69 — — Etoposide  3 mg/kg i.p 10 1018.7 25.7 1.33 ± 0.28 26.9 >0.05 Algal proteoglycan 20 mg/kg p.o 10 1018.7 27.8 1.75 ± 0.48 3.8 >0.05 Algal proteoglycan 30 mg/kg p.o 10 1018.6 27.5 1.44 ± 0.67 20.9 >0.05 Etoposide + 3 mg + i.p + p.o 10 10 18.724.2 1.21 ± 0.41 33.5 <0.05 Algal proteoglycan 20 mg/kg Etoposide + 3mg + i.p + p.o 10 10 18.6 23.4 1.17 ± 0.44 35.7 <0.01 Algal proteoglycan30 mg/kg

TABLE 18 Therapeutic Effect of Algal Proteoglycan Combined with5-Fluorouracil on S-180 Sarcoma in Mice Number of Body Weight TumorAnimals (g) Weight Tumor Route of in the in the in the in the (g)Inhibition Group Dose Administration Beginning End Beginning End x ± SDRate (%) P Value NS — 16 16 18.4 25.3 1.57 ± 0.51 — — 5-Fluorouracil 15mg/kg i.p 8 8 18.6 22.9 1.04 ± 0.52 33.8 <0.05 Algal proteoglycan 20mg/kg p.o 8 8 18.3 22.4 1.56 ± 0.51 0.6 >0.05 5-Fluorouracil + 15 mg +i.p + p.o 8 8 18.3 21.6 0.51 ± 0.28 67.5 <0.01 algal proteoglycan 20mg/kg

From the data listed in Tables 15-18, it can be seen that the algalproteoglycan at a dose of 20-30 mg/kg did not inhibit the growth ofS-180 sarcoma (P>0.05), and the algal proteoglycan at a dose of 60 mg/kginhibited growth of S-180 sarcoma with the inhibition rate of 35.4%,demonstrating an inhibitory effect of the algal proteoglycan on thegrowth of mouse sarcoma.

Carboplatin at a dose of 4 mg/kg used alone had no inhibitory effect onthe growth of the transplanted tumor of S-180 sarcoma in mice (P>0.05).When carboplatin at a dose of 4 mg/k was used in combination with 20mg/kg algal proteoglycan, the inhibition rate was 33%. Algalproteoglycan at a dose of 60 mg/kg used in combination with 7.5 mg/kgcyclophosphamide achieved an inhibition rate of 52.6% for the growth ofS-180 sarcoma, 10% higher than the inhibition rate achieved by usingcyclophosphamide alone.

The inhibitory effect on the growth of the transplanted tumor of S-180sarcoma in mice achieved by 3 mg/kg etoposide alone was 26.9% (P>0.05).When etoposide was used with 20 and 30 mg/kg algal proteoglycan, theinhibition rate was increased to 33.5% (P<0.05) and 35.7% (P<0.01)respectively.

5-fluorouracil at a dose of 15 mg/kg used alone had a moderateinhibitory effect on the transplanted tumor of S-180 sarcoma in mice.When 5-fluorouracil was combined with algae proteoglycan, the anti-tumoreffect was significantly increased with the inhibition rate of 67.5%,demonstrating that the combination of two drugs had produced asynergistic effect.

The above data showed that the combined use of the algal proteoglycanextract disclosed herein and chemotherapeutic agents such as5-fluorouracil, etoposide, carboplatin and cyclophosphamide can increasethe therapeutic efficacy of these chemotherapeutic drugs on S-180sarcoma in mice, and the synergistic effect of algal proteoglycancombined with 5-fluorouracil is more significant.

In addition, the applicants have also studied the inhibitory effect ofthe algal proteoglycan extract used alone and the extract used incombination with gemcitabine on in vivo tumor in nude mice withpancreatic cancer, as well as the inhibitory effect on pancreaticcarcinoma in patient-derived tumor xenograft models (PDX). The resultsshowed that the combined use of the algal proteoglycan extract disclosedherein and gemcitabine has also a synergistic effect on the treatment ofpancreatic carcinoma.

It is understood that, although the inventions has been described insome detail by way of illustration and examples, these inventions arenot limited to specific details described in these specific forms.Apparently to those skilled in this field, various equivalent changesand modifications to the technical features involved in the inventionsmay be practiced without deviating from the spirit of the inventionsdescribed herein, and such changes and modifications are within thescope of the inventions.

What is claimed is:
 1. A method for treating or preventing pain in asubject, comprising administering a therapeutically or prophylacticallyeffective amount of an algal proteoglycan extract or a compositioncomprising a therapeutically or prophylactically effective amount of thealgal proteoglycan extract to the subject.
 2. The method according toclaim 1, wherein the pain is tumor-associated pain.
 3. The methodaccording to claim 2, wherein the tumor is a primary tumor or ametastatic tumor.
 4. The method according to claim 3, the tumor isselected from sarcoma, lymphoma, esophageal cancer, pancreatic cancer,melanoma, leukemia, lung cancer, liver cancer, gastric cancer, breastcancer, cutaneous squamous carcinoma, colon cancer, or metastaticcancers thereof.
 5. The method according to claim 1, wherein the algalproteoglycan extract and/or the algal proteoglycan extract together witha pharmaceutically acceptable carrier are formulated intopharmaceutically acceptable dosage forms.
 6. The method according toclaim 5, wherein the pharmaceutically acceptable dosage forms areliquids for oral administration, capsules, powders, tablets, granules,pills, syrups or injectable formulations.
 7. The method according toclaim 1, wherein the algal proteoglycan extract is administered orally,subcutaneously, intramuscularly or intraperitoneally.
 8. The methodaccording to claim 1, wherein the algal proteoglycan extract isadministered orally.
 9. The method according to claim 1, wherein thealgal proteoglycan extract is administrated at a dosage of 5-500 mg/kgbody weight per day.
 10. The method according to claim 9, wherein thealgal proteoglycan extract is administrated at a dosage of 20-150 mg/kgbody weight per day.
 11. The method according to claim 10, wherein thealgal proteoglycan extract is administrated at a dosage of 30-120 mg/kgbody weight per day.
 12. The method according to claim 1, wherein thealgal proteoglycan extract is obtained by the following steps: (a)dissolving blue-green algae powder in a 5-20 fold volume of water, andbreaking the algal cell walls; (b) heating the resulting mixture in step(a) at 60-100 , and performing solid-liquid separation after cooling;(c) adjusting the pH of the resulting liquid in step (b) to less than 7,and performing solid-liquid separation again; and (d) adjusting theresulting liquid in step (c) to a neutral pH, concentrating the liquid,and drying the liquid if necessary.
 13. The method according to claim12, wherein the content of proteoglycan contained in the algalproteoglycan extract is more than about 50% by weight.
 14. The methodaccording to claim 12, wherein the content of proteoglycan contained inthe algal proteoglycan extract is more than about 60% by weight.
 15. Themethod according to claim 12, wherein the content of proteoglycancontained in the algal proteoglycan extract is more than about 65% byweight.
 16. The method according to claim 12, wherein the content ofproteoglycan contained in the algal proteoglycan extract is more thanabout 70% by weight.
 17. The method according to claim 12, wherein thecontent of proteoglycan contained in the algal proteoglycan extract ismore than about 75% by weight.